Fig 1: pMVP enables rapid, high-fidelity assembly of custom multicomponent transgene vectors. (A) The pMVP platform provides an array of Entry plasmids (pENTR) for promoters (6 options), 5′ modifiers (27 options), and 3′ modifiers (65 options) that can be partnered with a gene of interest and rapidly recombined into (B) an assortment of cross-compatible destination vectors (35 options) via an overnight recombination reaction to form (C) a customized experimental vector. In total, >108,000 unique vector permutations are possible for a gene of interest that is incorporated into pMVP. The resulting adenovirus (Ad), lentivirus (Lenti), Expression plasmid, Sleeping Beauty Transposon (SB), and PiggyBac transposon (PB) experimental vectors can be utilized in a broad variety of experimental contexts. (D) pMVP-derived vectors containing an EF1a promoter driving eGFP-P2A-PDX1-3xHA and either a polyA or WPRE were generated and added to primary (rat islet) or immortalized (HEK293) cells for either transient (adenovirus; expression) or stable (lentivirus, Sleeping Beauty, PiggyBac) transgene expression experiments. Cellular lysates were harvested for immunoblot analysis at the indicated times after viral transduction or plasmid transfection.
Fig 2: Targeting Sa-dCas9/LSD1 to PDX1 Area IV is sufficient to decrease PDX1 and disrupt PDX1-target gene expression. (A) UCSC genome browser screenshot of the PDX1 locus in the rat genome (rn5). Highlighted areas demark Area I-IV and gRNAArea IV targeting within Area IV. (B) Schematic of pMAGIC-derived adenoviruses expressing Sa-dCas9/LSD1. ChIP-qPCR analysis of (C) HA epitope tag and (D) H3K27ac enrichment using primers to a control region (Chr15) or Area IV in INS1 832/13 cells. Data are shown as a percent of input. (E) qRT-PCR analysis of PDX1 expression levels in INS1 832/13 cells. (F) Immunoblot analysis of INS1 832/13 cell lysates from 3 independent experiments. (G) qRT-PCR gene expression analysis in INS1 832/13 cells for genes directly regulated by PDX1. qRT-PCR analysis of (H) PDX1 and (I) the PDX1 target gene SLC2A2 (GLUT2) in intact primary rat islets. For all experiments, cells were harvested 48 h after transduction with either (–) gRNA control (white) or gRNAArea IV (black) purified adenoviruses depicted in panel B. Data represent mean ± S.E.M. of 3 (E, G) or 4 (C, D, H, I) independent experiments. #P < 0.05 compared to (–) gRNA control adenovirus.
Fig 3: Utilization of pMVP for the creation of unique PDX1 vectors. pENTR plasmids containing cDNA for human PDX1 with or without a stop codon (i.e. open) were recombined with pMVP components to generate an assortment of PDX1-expressing (A) adenovirus and (C) expression plasmid vectors. (B) Immunoblot blot analysis of INS1 832/13 cell lysates harvested 48h after treatment with crude adenovirus lysates. For the epitope tagged conditions, note the appearance of the endogenous (lower) and overexpressed (upper) PDX1 bands (top blot). For eGFP blot, the use of P2A adds 23 amino acids to the N-terminal protein (i.e. eGFP). Immunoblot analysis of HEK293 cell lysates 24 h after transfection of expression vectors encoding PDX1 with C-terminal (D) epitope tags, (E) eGFP reporter or fusion, (G) and mCherry reporter or fusion. (F, H) Fluorescence microscopy of the eGFP and mCherry containing conditions analyzed in panels E and G. Visible bands resulting from ‘Uncleaved’ P2A products (red circle •) and degradation products (*) are labeled.
Fig 4: Demonstration of pMVP inducible systems. (A) Graphical depiction of the mechanisms for the Tet-On, FKBP/ecDHFR degron domain, and AID systems and (B) the pMVP vectors generated and utilized. (C) Immunoblot analysis of INS1 832/13 cell lysates transduced with crude lysates for adenoviruses expressing PDX1 using either the Tet-On, ecDHFR, or FKBP systems and harvested at the indicated time intervals after addition (induction) and removal (washout) of the respective Dox, TMP, or Shld1 inducers. (D) Immunoblot analysis of INS1 832/13 cell lysates transduced with crude lysates for adenoviruses expressing PDX1 using either the Tet-On or ecDHFR systems and harvested 24h after addition of a dose curve of Dox (0.5–100 ng/ul) or TMP (1 nM–5 uM), respectively. (E) Immunoblot analysis of cell lysates from an INS1 832/13-derived cell line made by Sleeping Beauty-mediated integration of Tir1-P2A-AID-PDX1. Lysates were harvested at the indicated time intervals after treatment with 500uM auxin. (C–E) Endogenous PDX1 and PDX1-fusion bands were distinguished based upon size or the use of an epitope tag and are labeled accordingly.
Supplier Page from DNASU for PDX1 (Homo sapiens) in pENTR223.1 (Gateway donor/master vector)